Labeling Oligonucleotides Using T4 Polynucleotide Kinase

Tuesday, October 21, 2003

Description
Labeling Oligonucleotides Using T4 Polynucleotide Kinase

Procedure
1. Add the following to a microcentrifuge tube:
2 l of 10X Kinase Buffer
3 l of -[32P]-ATP (7000 Ci/mmole or 167 Ci/ul) (CAUTION! See Hint #1)
1 l of BSA
1 l of Oligonucleotide Primer (see Hint #2)
13 l of ddH2O
Add 30 Units of T4 Polynucleotide Kinase (USB)

2. Incubate at 37C for 60 min.

3. Add 80 l of TE Buffer.

4. Incubate for 5 min at 65C.

5. Poke two holes in the bottom of a 0.5 ml microcentrifuge tube with a 25-gauge needle.

6. Add approximately 1 mm of glass beads to hold the resin in the tube.

7. Add approximately 150 l of G10 resin (see Hint #3).

8. Place the 0.5 ml microcentrifuge containing glass beads and G10 resin inside of a 1.5 ml microcentrifuge tube.

9. Centrifuge for 1.5 min at setting "6" in a clinical centrifuge.

10. Discard the liquid from the bottom of the 1.5 ml microcentrifuge tube.

11. Repeat the centrifugation step and discard the liquid from the bottom of the 1.5 ml centrifuge tube.

12. Load the DNA sample onto the resin.

13. Centrifuge for 3 min at setting "6" in a clinical centrifuge.

14. Appropriately discard the column containing the nucleotides.

15. The labeled probe is in the eluate in the bottom of the 1.5 ml microcentrifuge tube.

16. Store at -20C.

Recipes
Oligonucleotide Primer 50 ng/ul of Oligonucleotide Primer
Requires between 40 to 50 ng oligonucleotide per reaction (per 1 ul)

TE Buffer 10 mM Tris
1 mM EDTA
pH 8.0

BSA 2 mg/ml Bovine Serum Albumin

Kinase Buffer (10X) Prepare just before use
10 l of 1 M MgCl2
10 l of 100 mM Spermidine
30 l of 0.5 M DTT
17 l of ddH2O
33 l of 2 M Tris, pH 7.6

0.5 M DTT

100 mM Spermidine

1 M MgCl2

2 M Tris, pH 7.6

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Labeling Oligonucleotides Using T4 Polynucleotide Kinase